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The Role of at Rich Interactive Domain 3A in the Tumorigenesis of Colorectal Carcinomas

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The Role of at Rich Interactive Domain 3A in the Tumorigenesis of Colorectal Carcinomas The role of AT rich interactive domain 3A in the tumorigenesis of colorectal carcinomas Meiying Song Department of Medical Science The Graduate School, Yonsei University The role of AT rich interactive domain 3A in the tumorigenesis of colorectal carcinomas Directed by Professor Hoguen Kim The Doctoral Dissertation submitted to the Department of Medicine, the Graduate School of Yonsei University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Meiying Song December 2012 ACKNOWLEDGEMENTS I would like to express my sincere gratitude to all those who made invaluable contributions to my research directly or indirectly. Without their invaluable helps and generous encouragements, this thesis could not have reached its present form. I am deeply indebted to my supervisor Professor Hoguen Kim, and Dr. Hyunki Kim working in Department of Pathology, Yonsei University. Their invaluable help, stimulating suggestion, constant encouragement, and patient guidance helped me in all the time of my research. My colleagues from Department of Pathology, Yonsei University also gave me lots of valuable advice when I was faced with problems during the process of my research and writing this thesis. I want to thank them for all their helps. Especially, I should give my thanks to my parents. It was their patient love and encouragement that enabled me to finish my doctorate in education at medical college of Yonsei University. TABLE OF CONTENTS ABSTRACT…….…………………………………………………………....1 I. INTRODUCTION.……………………………………………………….…..3 II. MATERIALS AND METHODS....……………………………….………...8 1. Patients and samples……………….………………………….…………...8 2. Tissue microarray and immunohistochemistry…………………….……...8 3. Evaluation of staining……………………………………………….……..9 4. Cell lines and cultures……………………………………………………..9 5. RNA extraction and semi-quantitative RT-PCR………………………......9 6. Real-time RT-PCR…………………………………………………….…10 7. Western blot……………….……………………………………………12 8. Plasmid construction……………………………………………….…...12 9. Small interfering RNA and transfection…………………………….…..13 10. Proliferation assay……………………………………………………..13 11. Cell cycle and apoptosis assay………………………………….….….14 12. Statistical analysis……………………………………………….…….15 III. RESULTS…………………………………………..…………………….16 1. Quantification and identification of ARID3A by proteomic analysis….16 2. Detection of ARID3A expression in colorectal cancer cell lines and tissues……………………………………………………………….….18 3. Immunohistochemical analysis of ARID3A expression in colorectal adenomas and carcinomas……………………………………………...21 4. Relationship between ARID3A expression and clinicopathological parameters……………………………….……………………..……….24 5. Evaluation of ARID3A as a potential prognostic marker for colorectal cancer………………………..…………………………………………26 6. Relationship between ARID3A expression and proliferation of colorectal cancer cell lines.………………………………………….…………….30 7. Relationship between ARID3A and CD133 expression………………..33 IV. DISCUSSION………………………………………………………….….37 V. CONCLUSION………………………………………………………......42 REFERENCES…………………………………………….………………….43 ABSTRACT (IN KOREAN)……………………………………………….48 PUBLICATION LIST……………………………………………………….50 LIST OF FIGURES Figure 1. Quantification and identification of ARID3A…….…....17 Figure 2. ARID3A expression in colorectal cancer cell lines…...18 Figure 3. ARID3A expression in colorectal cancer tissues………20 Figure 4. Representative images of ARID3A expression in three matched sets of normal, adenoma, and carcinoma tissues...………………………………………………...………22 Figure 5. Kaplan-Meier analysis of survival in patients with colorectal cancer…………………………….……..…...……27 Figure 6. Proliferation assay in RKO colon cancer cell line.........31 Figure 7. Upregulation of ARID3A expression in LoVo colon cancer cell lines.…………………………………………..….32 Figure 8. Overexpression of ARID3A reduces the transcription levels of pluripotency-associated markers and cancer stem cell markers………………………………………..…...34 Figure 9. ARID3A regulates the CD133 expression in colorectal cancer cells………………………….……………………...….36 LIST OF TABLES Table 1. Sequence of PCR primers…………………….………..11 Table 2. Clinicopathological features according to ARID3A expression in 690 colorectal cancers……………..……......25 Table 3. Relationship between clinicopathologic factors and overall survival by univariate Cox proportional hazards regression analysis……….……………………………………29 Table 4. Multivariate Cox regression analysis of overall survival...……………………...…………………………………30 ABSTRACT The role of AT rich interactive domain 3A in the tumorigenesis of colorectal carcinomas Meiying Song Department of Medical Science The Graduate School, Yonsei University (Directed by Professor Hoguen Kim) AT rich interactive domain 3A (ARID3A) is a member of the ARID family of DNA-binding proteins. Previous reports have shown that ARID3A controls the cell growth through p53-dependent manner. Recently, it has been reported that expression of ARID3A protein was 14.29-fold increased in colon cancer tissue, compared to matched normal colonic mucosa. To date, biological function of ARID3A in human disease, especially in colorectal cancer (CRC), remains largely unknown. Therefore, my research was focused on studying the role of ARID3A in the tumorigenesis and the physiological implication of CRC. The expression of ARID3A was investigated by immunohistochemistry. ARID3A expression was detected in a subset of colorectal adenomas and carcinomas, and the location of ARID3A was mainly in the nucleus. To investigate the prognostic impact of ARID3A in CRC, tissue specimens from 690 patients with CRC were examined. Of the 690 cases, 195 tumors were strong-positive for ARID3A, 187 tumors were weak-positive and 308 tumors were negative. The expression of ARID3A in CRC 1 was significantly correlated with age, degree of differentiation, depth of invasion, lymph node metastasis, distant metastasis, TNM stage, status of microsatellite instability, and CEA levels. The overall survival of CRC patients with ARID3A- strong expression was significantly longer than that of patients with ARID3A- negative or weak expression. On multivariate analysis, the strong expression of ARID3A was proven to be an independent predictor for better prognosis in CRC. Recent studies support a model in which ARID3A acts as a suppressor of lineage plasticity. I found that upregulation of ARID3A expression correlated with the reduction of transcriptional levels of several pluripotency-associated markers (OCT4, SOX2 and KLF4) and colorectal cancer stem cell (CSC) markers (CD133, CD44, CD166, CD24, and ALDH1) in CRC cell lines. Among these markers, I demonstrated the inverse relationship between ARID3A and CD133 expression in protein levels. In conclusion, my study strongly suggests that ARID3A might play an important role in colorectal carcinogenesis and can be used as a biomarker to predict the prognosis of CRC. Additionally, upregulation of ARID3A in CRC cell lines reduces expression levels of CD133, one of most important cancer stem cell markers in CRC. Further studies are necessary to delineate the mechanistic basis of these features. Key words: colorectal cancer, ARID3A, prognosis, cancer stem cell 2 The role of AT rich interactive domain 3A in the tumorigenesis of colorectal carcinomas Meiying Song Department of Medical Science The Graduate School, Yonsei University (Directed by Professor Hoguen Kim) I. INTRODUCTION Colorectal cancer (CRC) is the third most common cancer in the Western world. Every year, more than one million new cases are diagnosed as colorectal cancer worldwide, and colorectal cancer is also the third most common leading cause of cancer-related death in the developed countries 1. The classic description of colorectal carcinogenesis is the adenoma-carcinoma sequence. Most colorectal cancers develop through an ordered series of events beginning with the transformation of normal colonic epithelium to adenoma and then ultimately adenocarcinoma 2. Multiple genetic events are required for tumor progression, and genomic instability is recognized as an essential genetic feature that accompanies the acquisition of these mutations 3. Colorectal cancer is classified into at least three pathways of genomic and epigenomic instability: the chromosomal instability (CIN), microsatellite instability (MSI), and CpG island methylator phenotype (CIMP) pathways 3. Most colorectal cancers arise 3 sporadically. About 85% of sporadic cases have chromosomal instability; an allelic imbalance at some chromosomal loci (5q, 8q, 17p, and 18q), and chromosome amplification and translocation, which together contribute to tumor aneuploidy 1, 2, 4, 5. The remaining 15% of sporadic colorectal cancer have microsatellite instability- high (MSI-high) phenotypes 6. This phenotype is caused by mutation or loss of function through epigenetic gene silencing of DNA mismatch-repair genes 1, 7, 8. Prognostic markers are associated with survival, and predictive markers indicate likely benefit of treatment. Microsatellite instability and 18q imbalance are very important prognostic and predictive markers in colorectal cancer. These two markers can be used to discriminate between molecular subtypes in stage II CRC, which contribute to the risk-benefit assessment of adjuvant treatment 1. Even though cancer biomarkers, such as microsatellite instability and 18q imbalance, have been identified, the overall 5-year survival rate of colorectal cancer is still poor. Therefore, many efforts have been made to find other molecular markers to identify high-risk disease and to select patients for adjuvant treatment.
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